The publications and other materials referred to herein to illuminate the background of the invention and, in particular cases, to provide additional detail respecting its practice are incorporated herein by reference and, for convenience, are numerically referenced in the following text and respectively grouped in the appended bibliography.
Recombinant DNA Technology
With the advent of recombinant DNA technology, the controlled microbial production of an enormous variety of useful polypeptides has become possible. Already in hand are bacteria modified by this technology to permit the production of such polypeptide products such as somatostatin, the (component) A and B chains of human insulin, human growth hormone. More recently, recombinant DNA techniques have been used to occasion the bacterial production of proinsulin, thymosin alpha 1, (an immune potentiating substance produced by the thymus) and leukocyte interferon.
The workhorse of recombinant DNA technology is the plasmid, a non-chromosomal loop of double-stranded DNA found in bacteria and other microbes, oftentimes in multiple copies per cell. Included in the information encoded in the plasmid DNA is that required to reproduce the plasmid in daughter cells (i.e., a "replicon") and ordinarily, one or more selection characteristics such as, in the case of bacteria, resistance to antibiotics which permit clones of the host cell containing the plasmid of interest to be recognized and preferentially grown in selective media. The utility of plasmids lies in the fact that they can be specifically cleaved by one or another restriction endonuclease or "restriction enzyme", each of which recognizes a different site on the plasmidic DNA. Thereafter heterologous genes or gene fragments may be inserted into the plasmid by endwise joining at the cleavage site or at reconstructed ends adjacent to the cleavage site. DNA recombination is performed outside the cell, but the resulting "recombinant" plasmid can be introduced into it by a process known as transformation and large quantities of the heterologous gene-containing recombinant plasmid obtained by growing the transformant. Moreover, where the gene is properly inserted with reference to portions of the plasmid which govern the transcription and translation of the encoded DNA message, the resulting expression vehicle can be used to actually produce the polypeptide sequence for which the inserted gene codes, a process referred to as expression.
Expression is initiated in a region known as the promoter which is recognized by and bound by RNA polymerase. In some cases, as in the tryptophan or "trp" promoter preferred in the practice of the present invention, promoter regions are overlapped by "operator" regions to form a combined promoter-operator. Operators are DNA sequences which are recognized by so-called repressor proteins which serve to regulate the frequency of transcription initiation at a particular promoter. The polymerase travels along the DNA, transcribing the information contained in the coding strand from its 5' to 3' end into messenger RNA which is in turn translated into a polypeptide having the amino acid sequence for which the DNA codes. Each amino acid is encoded by a nucleotide triplet or "codon" within what may for present purposes be referred to as the "structural gene", i.e. that part which encodes the amino acid sequence of the expressed product. After binding to the promoter, the RNA polymerase first transcribes nucleotides encoding a ribosome binding site, then a translation initiation or "start" signal (ordinarily ATG, which in the resulting messenger RNA becomes AUG), then the nucleotide codons within the structural gene itself. So-called stop codons are transcribed at the end of the structural gene whereafter the polymerase may form an additional sequence of messenger RNA which, because of the presence of the stop signal, will remain untranslated by the ribosomes. Ribosomes bind to the binding site provided on the messenger RNA, in bacteria ordinarily as the mRNA is being formed, and themselves produce the encoded polypeptide, beginning at the translation start signal and ending at the previously mentioned stop signal. The desired product is produced if the sequences encoding the ribosome binding site are positioned properly with respect to the AUG initiator codon and if all remaining codons follow the initiator codon in phase. The resulting product may be obtained by lysing the host cell and recovering the product by appropriate purification from other bacterial protein.
Fibroblast Interferon
Human fibroblast interferon (FIF) is an antiviral protein which also exhibits a wide range of other biological activities (see ref. 1 for review). It has reportedly been purified to homogeneity as a single polypeptide of 19,000-20,000 molecular weight having a specific activity of 2 to 10.times.10.sup.8 units/mg (2,3). The sequence of the 13 NH.sub.2 -terminal amino acids of FIF has been determined (4). Houghton et al. (5) have used synthetic deoxyoligonucleotides (predicted from this amino acid sequence) to determine the sequence of the 276 5'-terminal nucleotides of FIF mRNA. Taniguchi et al. (6) and Derynck et al. (7) have recently employed RNA selection procedures to identify cloned cDNA copies of FIF mRNA in E. coli. See also Taniguchi et al., Gene 10, 11 (1980) and Proc. Natl. Acad. Sci. (U.S.A.) 77, 5230 (1980) and Nature 285, 547 (1980).
While isolation from donor fibroblasts has provided sufficient material for partial characterization and limited clinical studies with homogeneous fibroblast interferon, it is a totally inadequate source for the amounts of interferon heeded for large scale clinical trials and for broad scale prophylactic and/or therapeutic use thereafter. Indeed, presently clinical investigations employing human fibroblast-derived interferon in antitumor and antiviral testing have principally been confined to crude (&lt;1 percent pure) preparations of the material, and long lead times for the manufacture of sufficient quantities, even at unrealistic price levels, have critically delayed investigation on an expanded front.
We perceived that application of recombinant DNA technology would be the most effective way of providing large quantities of fibroblast interferon which, despite the absence in material so produced of the glycosylation characteristic of human-derived material, could be employed clinically in the treatment of a wide range of viral and neoplastic diseases.
More particularly, we proposed and have since succeeded in producing mature human fibroblast interferon microbially, by constructing a gene therefor which could then be inserted in microbial expression vehicles and expressed under the control of microbial gene regulatory controls.
Our approach to obtaining a fibroblast gene involved the following tasks:
1. Partial amino acid sequences would be obtained by characterization of fibroblast interferon purified to essential homogeneity, and sets of synthetic DNA probes constructed whose codons would, in the aggregate, represent all the possible combinations capable of encoding the partial amino acid sequences. PA1 2. Bacterial colony banks would be prepared containing cDNA from induced messenger RNA. The probes of part (1) would be used to prime the synthesis of radio-labelled single stranded cDNA for use as hybridization probes. The synthetic probes would hybridize with induced mRNA as template and be extended by reverse transcription to form induced, radio-labelled cDNA. Clones from the colony bank that hybridized to radio-labelled cDNA obtained in this manner would be investigated further to confirm the presence of a full-length interferon encoding gene. Any partial length putative gene fragment obtained would itself be used as a probe for the full-length gene. PA1 3. The full-length gene obtained above would be tailored, using synthetic DNA, to eliminate any leader sequence that might prevent microbial expression of the mature polypeptide and to permit appropriate positioning in an expression vehicle relative to start signals and the ribosome binding site of a microbial promoter. Expressed interferon would be purified to a point permitting confirmation of its character and determination of its activity notwithstanding the absence of glycosylation.